Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3K␣. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.The phosphoinositide 3-kinase (PI3K) 4 /Akt pathway is activated in a variety of solid and non-solid tumors (1) and therefore is considered as a potential intervention point for anticancer therapeutics. Activation of the pathway is frequently caused by mutations in PI3K␣ that enhance its catalytic activity, leading to the generation of phosphatidyl 3,4,5-trisphosphate (PIP3) (2) or by mutations or deletions in the tumor suppressor PTEN (phosphatase and tensin homolog) that result in its loss of function. PTEN antagonizes the activity of PI3K␣ through the dephosphorylation PIP3 (3). In addition, PI3K␣ can be activated by mutations in certain receptor-tyrosine kinases as well as by mutations in the oncogene KRAS (4, 5).The PIP3 generated by activation of PI3K␣ or sustained by the inactivation of PTEN binds to a subset of lipid-binding domains in downstream targets such as the pleckstrin homology (PH) domain of the oncogene Akt (6, 7); thereby, recruiting it to the plasma membrane. Once at the plasma membrane, Akt can be activated (8, 9). When active, Akt phosphorylates several effector molecules including the Forkhead box O (FOXO) transcription factors (10, 11). FOXO proteins are a family of conserved polypeptides that bind to DNA as a monomer and activate the transcription of genes that are involved in numerous biologically relevant processes such as metabolism, differentiation, proliferation, longevity, and apoptosis (12, 13). Akt phosphorylates FOXO proteins at three conserved consensus sites, which leads to conformational changes that facilitate CRM-1-mediated nuclear export (14, 15). Nuclear FOXO proteins function as regulators of transcription, whereas cytoplasmic FOXO proteins are considered inactive. It is well established that FOXO is negatively regulated by various proliferative and antiapoptotic signaling pathways that activate the PI3K/Akt signaling cascade (11). Therefore, we chose to employ a high content imaging approach to monitor the nucleocytoplasmic translocation of a GFP-FOXO3a fusion protein in U2OS cells (U2foxRELOC) (16,17) as the readout for biological inhibition...

Aplidin (plitidepsin) is a novel anticancer drug isolated from the marine tunicate Aplidium albicans. Aplidin shows potent antitumor activity in preclinical models against a wide variety of human tumors. Aplidin is currently in phase II clinical trials in a variety of solid tumors and hematologic malignancies. Moreover, clinical studies of Aplidin in combination with other agents are ongoing because it generally lacks cross-resistance with other known cytotoxic drugs. The mode of action of Aplidin in tumor cells is only partially understood. Aplidin induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor, the nonreceptor protein tyrosine kinase Src, and the serine threonine kinases c-Jun NH 2 -terminal kinase and p38 mitogen-activated protein kinase. Here, we show that sensitivity to Aplidin correlates inversely with the levels of expression of the cyclin-dependent kinase inhibitor p27 kip1 (p27) in a panel of low passaged human sarcoma cell lines. Aplidin induces p27 through an oxidation-dependent mechanism and the reduction of p27 levels by specific short hairpin RNA increases Aplidin sensitivity. We confirmed these results in p27 null mouse embryonic fibroblasts corroborating the specificity of the p27 role in Aplidin response because p21 waf1 null mouse embryonic fibroblasts do not show this increased sensitivity. We propose a mechanism of action of Aplidin involving p27 and support the analysis of p27 in the response to Aplidin in currently ongoing clinical trials to establish the levels of this protein as response predictor.

IntroductionThe activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells.MethodsWe used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation.ResultsWe defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment.ConclusionsThe constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0482-y) contains supplementary material, which is available to authorized users.

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.